Abnormal thrombotic events can give rise to heart attacks, stroke and deep vein thrombosis. One clotting disorder, Thrombotic Thrombocytopenic Purpura (TTP), is a thrombotic microangiopathy characterized by hemolytic anemia, consumptive thrombocytopenia, and ischemic injury. The pathogenesis of TTP is attributed to the presence of an Unusually Large von Willebrand Factor (ulvWF) multimers that lead to platelet clumping and subsequent thrombosis. vWF is primarily synthesized by vascular endothelial cells and secreted as a polymer with a Mr of greater than 500,000 kDa. These ultra-large multimers are highly active to promote platelet thrombi. Under normal physiological conditions, a metalloprotease enzyme, ADAMTS-13, cleaves vWF multimers into smaller protein units ranging from 500 to 20,000 kDa. Impairment of ADAMPTS-13 activity, caused either by hereditary deficiency or by acquired autoantibodies that specifically inhibit ADAMTS-13 function, leads to excessive accumulation of ulvWF and the eventual onset of TTP. The majority of clinically observed TTP cases in adults are acquired, with patients showing detectable levels of autoantibodies to ADAMTS-13.
Clinical management of TTP requires a rapid diagnosis followed with a prompt treatment. Plasma exchange therapy has been demonstrated to be the most successful therapy. It effectively replaces ADAMTS-13 and/or removes the ADAMTS-13 inhibitor in the case of acquired TTP. Without the prompt diagnosis and initiation of therapy, mortality for TTP is greater than 80%. However, timely treatment with plasma exchange greatly improves clinical outcome by inducing a remission in greater than 80% of patients. Therefore, a clinical test that can rapidly detect severe deficiencies of ADAMTS-13 activity associated with acute TTP is in great demand.
Since the discovery of ADAMTS-13 deficiency as a causal factor in TTP's pathogenesis, many attempts were made to measure plasma ADAMTS-13 activity in the patients with TTP. Furlan and Tsai independently reported original vWF protease assays. In both of their assays, human vWF, purified from plasma, was used as a substrate of ADAMTS-13 protease under a denatured condition using either urea or guanidine-HCl. After incubation with the tested plasma sample, ADAMPTS-13 activities were estimated using electrophoresis/Western blot that detect either vWF substrate disappearance or generation of vWF cleavage products. The assays are tedious, technically challenging, and difficult to standardize. Other diagnostic tests have also been reported since the original works by Furlan and Tsai. They include collagen binding assays, immunoradiometric assay and ristocetin cofactor activity assays. Although these assay methods require less time to complete, they do not directly measure the vWF cleavage by ADAMTS-13 protease.
Recently, a recombinant vWF protein containing a GST fusion protein and a Histidine tag (vWF73) was developed. This substrate contains a specific cleavage site for ADAMTS-13 (Tyr1605-Met1606) and has been used to measure ADAMTS-13′ activity in TTP patients. The assay demonstrated excellent reproducibility but the test turnaround time is at least 24 hours because it utilizes a Western blot to detect the cleavage product.
Therefore there is a need for a rapid, easy to perform, and functionally relevant ADAMTS-13 assay to provide clinical data for diagnosis and effective management of patients suspected to have TTP.